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Separating and screening method for lytic aquatic pathogenic bacterium phage

A phage and aquatic technology, applied in the biological field, can solve problems such as the difficulty of separating phages and the uncertainty of phage distribution, and achieve the effect of avoiding or reducing the use

Active Publication Date: 2014-10-01
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although phages are widely found in various environments in nature, the distribution of phages is uncertain
In many cases, it is difficult to isolate phages by conventional methods. This situation occurs in the isolation of phages from various pathogenic bacteria.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0039] ⑷Preparation of host bacterial genome DNA

[0040]Inoculate the host bacteria in LB liquid medium, shake culture at 37°C overnight, discard the supernatant by centrifugation, add TE buffer to the pellet to resuspend the bacteria, add lysozyme and place at room temperature for 10 minutes, add SDS and proteinase K to mix well Afterwards, keep warm in a water bath at 60°C for 6 hours, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), shake and mix well, centrifuge to take the supernatant in a new centrifuge tube, and extract until there is no protein film Add chloroform to the supernatant for extraction, take the supernatant in a new centrifuge tube, add 2 times the volume of 95% ethanol (pre-cooled) to the supernatant, shake and mix, centrifuge and discard the supernatant; Add 70% ethanol, centrifuge at 10000rpm for 5min, discard the supernatant, and dry at room temperature; add double distilled water or TE buffer solution (pH8.0) to obtain the host ba...

Embodiment 1

[0050] According to the above technical scheme, a phage capable of lysing Aeromonas vernerii and a corresponding host strain of Aeromonas vervetii were isolated. The name of the bacteriophage is ΦA8-29, the classification name is Aeromonas veronii phage, the deposit number: CGMCC No.9152; the name of Aeromonas veronii is A8-29, the classification name is Aeromonas veronii, the deposit number: CGMCC No.9093, the depository unit : General Microbiology Center of China Microbiological Culture Collection Management Committee, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation date: April 25, 2014.

[0051] (1) Specimens: collect samples of aquatic products such as freshwater fish of different species.

[0052] (2) Specimen processing: collect the gastrointestinal contents of the fish, put them in a 1.5ml centrifuge tube, and store them at 4°C.

[0053] (3) Isolation of aquatic pathogenic bacteria: Mix the contents of the gastrointestinal tract, and...

Embodiment 2

[0061] Adopt the same technical plan and steps to collect different aquatic product samples, and isolate the phage ФA33 of Aeromonas temperii from the samples. The titer or titer of the phage is 3.0×10 9 pfu / ml.

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Abstract

The invention relates to a separating and screening method for a lytic aquatic pathogenic bacterium phage. The name of the phage is deltaA8-29, the systematic name is Aeromonas veronii phage, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.9152; the name of Aeromonas veronii is A8-29, the systematic name is Aeromonas veronii phage, the preservation number is CGMCC No.9093, the preservation unit is CGMCC, the address is #3, yard 1, West Beichen Road, Chaoyang District, Beijing, and the preservation date is April 25, 2014. The invention provides the screening and identification method of the aquatic pathogenic bacterium phage. The separated phage can be used for preventing and controlling infection of aquatic pathogenic bacteria and avoiding or reducing use of antibiotics in aquaculture so as to develop the green mode of aquaculture.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for separating and screening phages of aquatic pathogenic bacteria. Background technique [0002] my country's national aquaculture area in 2007 was 5633.21kha, and the aquaculture output was 47.475 million tons, accounting for more than 2 / 3 of the world's total aquaculture output, and the aquaculture output exceeded the fishing output. Diseases occur on about 1 / 10 of the aquaculture area every year, and the annual loss of aquaculture production due to diseases accounts for about 30%, and the direct economic loss exceeds 10 billion yuan. Among them, the incidence and fatality rate of bacterial fish diseases are close to or exceed 50%. [0003] Important aquatic pathogens include Vibrio, Aeromonas, Pseudomonas, Flavobacterium, Acinetobacter, Pseudomonas, Pasteurella, Edwardsiella, Edwardsiella tarda, Yersinia, Serratia, Staphylococcus, Streptococcus, Nocardia, Clostridium and ...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/02C12R1/92
Inventor 杨洪江何洋
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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